Anti-inflammatory use of liquid phytocomplexes from olive

ABSTRACT

The present invention relates to a phytocomplex or natural concentrate rich in polyphenolic compounds, such as hydroxytyrosol and 3,4-DHPA-EDA, derived from the vegetation waters of oil-bearing olives or from olive pomace resulting from the olive milling process for use in the treatment and prevention of angiogenesis and inflammation. In particular, the angiogenesis and inflammation to which reference is made is pathologic angiogenesis and inflammation, for example that which sustains the development and spread of a tumor or angiogenesis and inflammation tied to non-tumor pathologies. The present invention further relates to a beverage comprising the polyphenol concentrate and the use thereof in the treatment and prevention of angiogenesis and inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage application of InternationalPCT Application PCT/IB2014/065747, filed Oct. 31, 2014, which claimsbenefit of priority to Italian Patent Application No. MI2013A001815,filed Oct. 31, 2013, both of which are incorporated herein by referencein their entirety.

The present invention relates to the use of a natural phytocomplex richin polyphenolic compounds, in particular rich in hydroxytyrosol and3,4-DHPA-EDA, derived from the water resulting from the pressing ofoil-bearing olives (commonly known as vegetation water) or from residualolive pomace resulting from the olive milling process in the preventionand treatment of angiogenesis and/or inflammation.

In particular, the angiogenesis and inflammation to which reference ismade is of a pathological type, for example that which sustains thedevelopment and spread of a tumor, or the angiogenesis and inflammationtied to non-tumor pathologies.

Angiogenesis is a physiological process which occurs during the stagesof growth and development of an individual and it involves the formationof new blood vessels from those of the pre-existing vascularcompartment.

Angiogenesis is a process which also characterizes various pathologicalphenomena, including tumors.

A tumor (or neoplastic) mass can grow and develop autonomously up to asize of around 1-2 mm³; however, in order to grow further, it mustassure itself a vital supply of nutrients and oxygen and it thereforeneeds to create its own vascular compartment.

A tumor is capable secreting angiogenic factors, such as VEGF (VascularEndothelial Growth Factor), bFGF (basic Fibroblast Growth Factor) andPDGF (Platelet-Derived Growth Factor), which are capable of promotingthe development of blood vessels. The angiogenic factors secreted by thetumor activate endothelial cells, which in response begin to proliferateand secrete substances which degrade the cellular matrix and basementmembranes (e.g. matrix metalloproteases-MMP) in order to migrate andinvade the surrounding tissues. Subsequently, the endothelial cellsorganize to form tubular structures stabilized by the presence ofpericytes, i.e. contractile cells which surround the newly formed bloodvessels and are capable of modifying blood flow and of regulating vesselpermeability. Blood vessels deriving from tumors are irregular and arecharacterized by structural elements that distinguish them from “normal”vessels. For example, they are characterized by the absence ofpericytes, large fenestrae and pronounced vessel dilation. Thesecharacteristics alter the permeability and pressure levels of thevessels and consequently also interfere with the delivery of anti-tumordrugs, which, instead of reaching the tumor, are dispersed ininterstitial liquid and are thus unable to perform their function.

Considering the importance of angiogenesis in the processes ofdevelopment, growth and metastatization of tumors, numerous studies havebeen conducted with the aim of identifying substances that are capableof blocking the irregular development of tumor blood vessels and arethus able to improve the delivery of drugs to the tumor site. Inparticular, large efforts have been made to identify molecules capableof preventing the anomalous development of blood vessels; this has ledto the formulation of the concept of “angioprevention” (i.e. theprevention of tumor-related angiogenesis).

The majority of these molecules are of natural origin (or are in anycase synthetic analogues). A very interesting example is moleculesderiving from olive oil.

It is well known, in fact, that the incidence rates of cardiovascularpathologies and tumors are significantly lower in populations that adoptthe Mediterranean diet, which is based on the consumption of olive oil.The scientific evidence has provided a considerable incentive to thestudy of olive oil with the aim of defining its composition and, inparticular, of identifying substances with a medical-pharmacologicalpotential.

One characteristic of olive oil which has aroused particular interest isthe high level of polyphenols contained in it. These compounds arenatural antioxidants of plant origin which are capable of inhibiting theformation of free radicals.

The beneficial properties of olive oil have induced a considerableincrease, above all in Italy, in olive cultivation and olive oilproduction. As a consequence, we have also witnessed a strong increasein by-products of olive oil production, mainly vegetation water andpomace, which are highly polluting and thus generate a considerableenvironmental impact.

The disposal of this material is strictly regulated in Italy on both anational and regional level and the implementation of legislation (law574 of 11/1996) results in hefty costs for producers, who are unable toderive any advantage from these waste products, which, however, are richin molecules with a high medical and pharmaceutical potential.

Hydroxytyrosol is the polyphenol contained in vegetation water that hasbeen most studied. It is present in vegetation water and pomace and isformed also by hydrolysis of oleuropein, a substance that is presentabove all in the leaves of olive trees.

Recent research has demonstrated that hydroxytyrosol on its own has acytoprotective effect against PC12 cells (a pheochromocytoma cell line),is anti-apoptotic when administered to U937 cells (a humanmyelomonocytic line) and C2C12 cells (a mouse myoblast line), inhibitsbreast tumor proliferation in vivo in the case of induced neoplasias, isa chemopreventive agent in studies on HL60 and HL60R tumor cell lines (ahuman promyelocytic leukemia line and its multi-drug resistantderivative) and is a preventive agent against premenstrual syndrome andosteoporosis.

Moreover, it has been demonstrated that in vivo administration ofhydroxytyrosol (also in high concentrations) has no toxic effect.

Other research has demonstrated that when oleuropein is administered onits own, it performs an anti-microbial activity, has an anti-tumorpotential in colorectal tumor cell lines, metastatic breast tumors andER-negative cell lines and has the ability to alter cellular stabilityon a cytoskeletal level.

Though many studies have been undertaken on vegetation water, there isstill a greatly felt need to identify new properties which can lendvalue to this waste product, which would otherwise be only a cost forthe producer and a hazard to the environment. There is a particularlyfelt need to identify new nutritional and medical-pharmacologicalproperties which may dignify this waste product.

In this regard, the Applicant has surprisingly found that vegetationwater is capable of blocking/preventing, both in vitro and in vivo,angiogenesis and inflammation, in particular pathologic angiogenesis andinflammation, for example that which sustains the development and spreadof a tumor, or the angiogenesis and inflammation associated withnon-tumor pathologies. In particular, the Applicant has found that byconcentrating, via reverse osmosis, the permeate of vegetation waterssubjected to microfiltration, one obtains a phytocomplex rich inpolyphenolic compounds capable of preventing and blocking angiogenesisand inflammation, in particular pathologic angiogenesis andinflammation, such as that/those associated with a tumor or that/thoseassociated with a non-tumor pathology, in a manner that is moreeffective compared to what the same compounds are capable of achievingwhen taken individually, i.e. isolated from vegetation waters and pomaceby means of purification techniques.

This effect is particularly advantageous for human health, above all interms of angioprevention. In fact, to this end the vegetation waterconcentrate of the present invention, on its own or in association withfurther anti-tumor and anti-angiogenic and anti-inflammatory substances,can be used, for example in the form of a beverage, to treat or preventangiogenesis and inflammation, in particular pathologic angiogenesis andinflammation associated with a tumor or pathologic angiogenesis andinflammation associated with a non-tumor disease.

Further advantages of the present invention will be apparent from thedetailed description that follows, which is made with the aid of theappended Figures, in which:

FIG. 1 shows the results of the MTT assay aimed at evaluating theproliferation of HUVE cells following treatment with progressivedilutions of: A) the polyphenol concentrate of the present invention(sample A009); B) the blank (sample A012); C) purified hydroxytyrosol(HyT); and D) the purified hydroxytyrosol blank (i.e. ethanol-EtOH).

FIG. 2 shows the results of the assay on apoptosis and HUVE cell deathfollowing treatment with progressive dilutions of: A,C) the polyphenolconcentrate of the present invention (sample A009); B,D the blank(sample A012) 24 and 48 hours after treatment.

FIG. 3 shows the results of the assay on apoptosis and HUVE cell deathfollowing treatment with progressive dilutions of: A,C) purifiedhydroxytyrosol (HyT); B,D the purified hydroxytyrosol blank (i.e.ethanol-EtOH) 24 and 48 hours after treatment.

FIG. 4 shows the results of the morphogenesis assay based on anevaluation of the ability of HUVE cells to form capillary-type tubularstructures in matrigel following treatment with: A) serum-free culturemedium (SFM), complete culture medium (CTRL), 1:500 and 1:250 dilutionsof the polyphenol concentrate of the present invention (sample A009),1:500 and 1:250 dilutions of the blank (sample A012); B) serum-freeculture medium (SFM), complete culture medium (CTRL), 1:500 and 1:250dilutions of purified hydroxytyrosol (HyT), 1:500 and 1:250 dilutions ofthe purified hydroxytyrosol blank (i.e. ethanol-EtOH).

FIG. 5 shows the results of the chemotaxis assay on HUVE cells followingtreatment with: A) complete culture medium (CTRL), serum-free culturemedium (SFM), progressive dilutions of the polyphenol concentrate of thepresent invention (sample A009), the blank (sample A012); B) completeculture medium (C+), serum-free culture medium (SFM), progressivedilutions of purified hydroxytyrosol (HyT) and of the purifiedhydroxytyrosol blank (i.e. ethanol-EtOH).

FIG. 6 shows the results of the chemoinvasion assay on the HUVE cellsfollowing treatment with: A) complete culture medium (CTRL), serum-freeculture medium (SFM), progressive dilutions of the polyphenolconcentrate of the present invention (sample A009), the blank (sampleA012); B) complete culture medium (C+), serum-free culture medium (C−),progressive dilutions of purified hydroxytyrosol (HyT) and of thepurified hydroxytyrosol blank (i.e. ethanol-EtOH).

FIG. 7 shows the results for the oxidative stress (measured as % ofDCFH-DA⁺ cells) affecting HUVE cells treated with H₂O₂ prior totreatment with A) progressive dilutions of the polyphenol concentrate ofthe present invention (sample A009) and respective blank (sample A012);B) progressive dilutions of purified hydroxytyrosol (HyT) and therespective blank (i.e. ethanol-Eton).

FIG. 8 shows the results for the oxidative stress (measured as % ofDCFH-DA⁺ cells) affecting HUVE cells treated with H₂O₂ followingpre-treatment with A) progressive dilutions of the polyphenolconcentrate of the present invention (sample A009) and respective blank(sample A012); B) progressive dilutions of purified hydroxytyrosol (HyT)and the respective blank (i.e. ethanol-EtOH).

FIG. 9 shows the results of the macroscopic colorimetric analysis ofmatrigel inocula implanted beneath the skin of mice A) without treatment(matrigel alone), in the presence of VTH-VEGF,TGF,HGF (positive controlC+), in the presence of VTH and a 1:500 dilution of the polyphenolconcentrate of the present invention (sample A009) and the respectiveblank (sample A012); B) without treatment (matrigel alone), in thepresence of VTH-VEGF,TGF,HGF (positive control C+), in the presence ofVTH and a 1:500 dilution of purified hydroxytyrosol (HyT).

FIG. 10 shows the assay of the hemoglobin in the explanted matrigelinocula of FIG. 9.

The present invention relates to a phytocomplex or concentrate ofvegetation waters and pomace comprising polyphenolic compounds,preferably hydroxytyrosol and 3,4-DHPA-EDA, for use in the treatment andprevention of angiogenesis and inflammation.

Preferably, the angiogenesis and inflammation to which reference is madeis pathologic angiogenesis and inflammation, more preferablytumor-related angiogenesis and inflammation. Alternatively, theangiogenesis and inflammation may be associated not with a tumor butrather pathologies such as: rheumatic diseases, preferably rheumatoidarthritis and gout; inflammatory diseases of the colon-rectum,preferably Crohn's disease, irritable and ulcerative bowel syndrome;bronchial pathologies, preferably bronchus chronic obstructive pulmonarydisease and asthma; liver diseases preferably cirrhosis and fibrosis;diseases of the prostate, preferably prostatic hyperplasia andacute/chronic prostatitis; mucositis; dermatitis or pre-neoplasticlesions, e.g. breast, uterus, lung or mouth lesions.

In fact, the concentrate of vegetation waters and/or olive pomace of thepresent invention surprisingly demonstrated to be particularly effectivein preventing the neo-formation of tumor blood vessels (i.e.angiogenesis). These vessels have a distinctive physicochemicalstructure and hence biological functioning compared to normal bloodvessels. They are largely fenestrated and often devoid of pericytes andfor this reason have an altered vessel permeability. This structure isoften at the basis of the failure or difficulties of manypharmacological treatments, because the drug is lost in interstitialspaces during delivery to the tumor through the vessels and thus eitherfails to reach the target or reaches it in ineffective quantities.Inhibiting the formation of this type of vessel structure clearlyenables better delivery of the substances to the tumor and hence thepossibility of improving the tumor treatment.

The vegetation waters preferably derive from an olive milling processwith three phases (oil, vegetation waters and pomace), and two phases(oil and pomace+vegetation waters). Preferably, the vegetation watersgenerated by the mill can be treated with a solution with an acidic pH,preferably a pH ranging from 3 to 5, preferably 4-5, for example,through the addition of a strong acid, and with pectolytic enzymes, i.e.enzymes that hydrolyze the cellulose matrix of the olive skin.

The pomace is preferably pitted, diluted and pre-filtered. The pomacepreferably has a size or cut-off ranging from 0.5 to 1 millimeter (mm),more preferably about 0.7 mm. An example of a cut-off is the oneobtained with a vibration screen.

If necessary, the pitted pomace can be solubilized or dispersed in anaqueous matrix with a pH comprised from 3 to 5, preferably from 3.5 to4.

The solubilization step has the purpose of solubilizing the polyphenolswhich would otherwise remain trapped in the solid matrix of the oliveskins. In a preferred embodiment of the invention, the concentrate ofvegetation waters and/or olive pomace (hereinafter “the concentrate”)further comprises: at least a further phenolic compound preferablyselected from: tyrosol, chlorogenic acid, β-hydroxyverbascoside, rutin,verbascoside and luteolin; and at least one metal preferably selectedfrom: sodium, calcium, magnesium and potassium; and at least an anionpreferably selected from: chlorides, sulphates, phosphates and nitrates;and at least one glucide selected from: glucose, fructose, mannitol andsucrose.

In a further embodiment of the invention, the concentrate comprisesnitrogenous substances (proteins, amino acids), preferably in an amountcomprised from 15 to 60 mg/kg, more preferably from 20 to 40 mg/kg (mgof nitrogen per liter of active solution). In any case the phenoliccompounds present in the concentrate in the largest amount arehydroxytyrosol and 3,4-DHPA-EDA.

Preferably, the amount of the hydroxytyrosol ranges between 1 and 10grams per liter of vegetation waters (g/L), more preferably between 1.5and 5 g/L, even more preferably between 2 and 3 g/L.

Preferably, the amount of 4-DHPA-EDA is comprised from 0.5 to 8 g/L,more preferably from 1 and 6 g/L, even more preferably from 1.5 to 2.5g/L.

Preferably, the amount of tyrosol is comprised from 0.1 to 0.4 g/L, morepreferably from 0.15 g/L to 0.25 g/L.

Preferably, the amount of chlorogenic acid is comprised from 0.06 to0.24 g/L, more preferably from 0.8 to 0.16 g/L.

Preferably, the amount of β-hydroxyverbascoside is comprised from 0.3 to1.5, more preferably from 0.5 to 1 g/L.

Preferably, the amount of rutin is comprised from 0.05 to 0.2 g/L, morepreferably from 0.08 to 0.15 g/L.

Preferably, the amount of verbascoside is comprised from 0.4 to 1.7 g/L,more preferably from 0.6 to 1 g/L.

Preferably, the amount of luteolin is comprised from 0.1 to 0.5 g/L,more preferably from 0.15 to 0.28 g/L.

Preferably, the amount of sodium is comprised from 75 to 300 mg/L, morepreferably from 120 to 180 mg/L.

Preferably, the amount of calcium is comprised from 5 to 10 g/L, morepreferably from 2 to 5 g/L.

Preferably, the amount of magnesium is comprised from 220 to 900 mg/L,more preferably from 400 to 500 mg/L.

Preferably, the amount of potassium is comprised from 3 to 15 g/L, morepreferably from 6 to 9 g/L.

Preferably, the amount of chlorides is comprised from 1.5 to 7 g/L, morepreferably from 2.5 to 4.5 g/L.

Preferably, the amount of sulphates is comprised from 12 to 45 g/L, morepreferably from 18 to 28 g/L.

Preferably, the amount of phosphates is comprised from 1.5 to 7 g/L,more preferably from 2.5 to 5 g/L.

Preferably, the amount of nitrates is comprised from 12 to 50 mg/L, morepreferably from 18 to 30 mg/L.

Preferably, the amount of glucose is comprised from 15 to 60 g/L, morepreferably from 25 to 35 g/L.

Preferably, the amount of fructose is comprised from 3.5 to 15 g/L, morepreferably from 5 to 9 g/L.

Preferably, the amount of mannitol is comprised from 1 to 4 g/L, morepreferably from 1.5 to 3 g/L.

Preferably, the amount of sucrose is comprised from 4 to 16 g/L, morepreferably from 6 to 10 g/L.

In a preferred embodiment of the invention, the concentrate isobtained/obtainable by means of a process comprising the steps: (i)microfiltering a sample of the vegetation water and/or olive pomace soas to obtain a concentrate and a permeate of microfiltration; and (ii)concentrating by reverse osmosis the microfiltration permeate of step(i). Preferably, the microfiltration takes places after thesolubilization step as described earlier.

The microfiltration has the purpose of separating a concentrate, thatis, the concentrated fraction of the content in suspension of thevegetation waters/pomace, for example micro-fragments, fibers andcorpuscular material such as cells and bacteria. It is carried out underthe standard conditions for this type of matrix.

In addition to the concentrate, following the microfiltration step oneobtains a permeate, i.e. a clear fraction, characterized by a color thatvaries according to the starting material and which contains thedissolved components of the vegetation waters/pomace, e.g. proteins,sugars, salts, polyphenols, organic acids and various soluble organicmolecules.

Preferably, the microfiltration is carried out with at least one andpreferably two ceramic membrane(s). The membrane is preferablycharacterized by a tubular shape.

In a preferred embodiment, the membrane is made from aluminum oxide andzirconia.

Preferably, the membrane has the following characteristics: an outerdiameter ranging from about 30 to about 40 mm, preferably about 25 mm;and a length ranging from about 500 to about 1500 mm, preferably about1200 mm; and a series of channels with a diameter, preferably ahydraulic diameter, ranging from about 2.5 to about 5 mm, preferablyabout 3.5 mm; and a filtering surface ranging from about 0.15 to about0.7 m², preferably about 0.35 m²; and a molecular cut-off ranging fromabout 0.1 micron to about 300 kDa.

The reverse osmosis step for concentrating the permeate obtained fromthe microfiltration of the vegetation waters/pomace as earlierdescribed, is carried out under the standard conditions for this type ofmatrix, preferably by using a polymeric membrane, more preferably madeof polyamide.

In particular, the membrane has a wound spiral shape and a molecularcut-off with a high salt rejection, that is, capable of rejecting sodiumchloride molecules at a percentage of 99.9%. This means that the osmosismembrane traps the molecules of biomedical interest and allows onlywater molecules to pass through.

Preferably, the polymeric membrane has a filtering surface which rangesfrom about 5 to about 15 m², and is more preferably about 7 m².

The reverse osmosis step serves to concentrate the permeate obtainedfrom the microfiltration preferably by about 4 times; this means thatfrom 100 L of microfiltration permeate one will obtain 25 L ofconcentrate.

In this case the Volume Concentration Ratio (VCR) is 4, i.e. 100/25.

The VCR can change based on the starting matrix (vegetation water) andabove all based on its salt content, because the process of reverseosmosis must counterbalance the osmotic pressure of the matrix that isbeing concentrated.

The present invention further relates to a concentrate (or phytocomplex)of vegetation waters/pomace that is obtainable/obtained with theabove-described process.

The concentrate preferably has the composition earlier described inrelation to the content of phenolic compounds, metals, carbohydrates,anions and nitrogen. It can be used on its own or in combination withother substances, molecules or anti-tumor and anti-angiogenic andanti-inflammatory therapies for the treatment and prevention ofangiogenesis and inflammation, preferably pathologic angiogenesis andinflammation, in particular angiogenesis and inflammation associatedwith tumors or angiogenesis and inflammation associated with non-tumorpathologies such as: rheumatic diseases, preferably rheumatoid arthritisand gout; inflammatory diseases of the colon-rectum, preferably Crohn'sdisease, irritable and ulcerative bowel syndrome; bronchial pathologies,preferably bronchus chronic obstructive pulmonary disease and asthma;liver diseases preferably cirrhosis and fibrosis; diseases of theprostate, preferably prostatic hyperplasia and acute/chronicprostatitis; mucositis; dermatitis or pre-neoplastic lesions, e.g.breast, uterus, lung or mouth lesions. Preferably, the concentrate ofthe present invention can be used on its own or in combination withother substances/molecules, with the aim of inhibiting, preferably in apreventive manner, the formation of tumor blood vessels.

The tumors to which the present invention makes reference are preferablycolorectal, breast and prostate cancer, skin cancers (melanoma andothers), cancers of the pancreas, lungs, ovaries, bladder, kidneys andliver.

The inflammatory conditions associated with the angiogenesis to whichthe present invention makes reference are: rheumatic diseases,preferably rheumatoid arthritis and gout; inflammatory diseases of thecolon-rectum, preferably Crohn's disease, irritable and ulcerative bowelsyndrome; bronchial pathologies, preferably bronchus chronic obstructivepulmonary disease and asthma; liver diseases preferably cirrhosis andfibrosis; diseases of the prostate, preferably prostatic hyperplasia andacute/chronic prostatitis; mucositis; dermatitis or pre-neoplasticlesions, e.g. breast, uterus, lung or mouth lesions.

A further aspect of the present invention relates to a beveragecomprising the concentrate of vegetation waters/pomace earlier describedand possible excipients normally added for the production of varioustypes of beverages.

The beverage can be based on water and fruit and milk. In theparticularly preferred embodiment of the invention, the beverage isbased on fruit, preferably it is a based on grape juice. Preferred inparticular are grape juice and grape must, preferably from organicgrapes. The beverage can optionally be lyophilized.

Alternatively, the concentrate of vegetation waters/pomace earlierdescribed can be formulated as pills, lozenges or tablets for oral use.

Practically speaking, the beverage or oral formulation can be taken as afood supplement, in particular with the aim of preventing angiogenesisand inflammation, preferably pathologic angiogenesis and inflammation,in particular that associated with a tumor or that associated with anon-tumor pathology such as: rheumatic diseases, preferably rheumatoidarthritis and gout; inflammatory diseases of the colon-rectum,preferably Crohn's disease, irritable and ulcerative bowel syndrome;bronchial pathologies, preferably bronchus chronic obstructive pulmonarydisease and asthma; liver diseases preferably cirrhosis and fibrosis;diseases of the prostate, preferably prostatic hyperplasia andacute/chronic prostatitis; mucositis; dermatitis or pre-neoplasticlesions, e.g. breast, uterus, lung or mouth lesions.

Preferably, the beverage or oral formulation is taken as a foodsupplement with the aim of inhibiting, preferably in a preventivemanner, the formation of tumor blood vessels.

Optionally, the beverage can be taken in association with anti-tumor andanti-angiogenic and anti-inflammatory substances, molecules, drugs ortherapies.

A further aspect of the present invention relates to a cream, oil,ointment, mist, shampoo or gel comprising the concentrate of vegetationwaters/pomace earlier described and possible excipients.

Said cream, oil, ointment or gel can be used for the treatment,preferably topical, and/or the prevention of a physiopathologicalcondition caused by an increased and/or altered angiogenesis and byinflammation.

EXAMPLE Production of Concentrated Polyphenols from Olive VegetationWaters/Pomace

The entire production process is centered on the use of membrane-basedtangential-flow filtration and separation technologies.

The membrane process carried out for the production of polyphenolconcentrates from olive vegetation waters/pomace used only twofiltration stages: microfiltration and reverse osmosis. However,depending on the product desired it is possible to complete the processwith ultrafiltration and nanofiltration stages.

Microfiltration enables the separation of suspended solids, bacteria andfats, whilst reverse osmosis traps all the substances present, includingions, also monovalent ones, and allows only water to permeate.

The process was carried out starting from vegetation waters derivingfrom an olive milling process with three phases, but it can also beapplied to wet pomace deriving from a two-phase process after apre-treatment.

The wet pomace can be pitted, diluted and pre-filtered with a cut-off ofabout 0.7 mm (for example with a vibrating screen) and then treated withmembrane systems; or else it can be treated in a three-phase decanterwith possible dilutions and reprocessing in a three-phase decanterbefore being treated with membrane processes.

The microfiltration process carried out has the objective of separatingthe concentrated fraction of the entire contents in suspension(micro-fragments, fibers and corpuscular material such as cells andbacteria) present in the vegetation waters/pomace.

The microfiltration permeate is a clear fraction whose color differsaccording to the cultivar of the treated olives and which contains allof the dissolved components of the vegetation waters/pomace, e.g.proteins, sugars, salts, polyphenols, organic acids and various solubleorganic molecules.

For the microfiltration, use was made of two tubular aluminum oxideceramic membranes with a selective layer of zirconium oxide having thefollowing characteristics: outer diameter 25 mm, length 1178 mm, 23channels with a channel hydraulic diameter of 3.5 mm, filtering surfaceof 0.35 m² and molecular cut-off respectively of 0.14 micron-300 KDa.

In microfiltration, by using membranes with 23 channels having ahydraulic diameter of 3.5 mm, it is possible to concentrate thevegetation waters/pomace until obtaining a concentrated fraction with atotal solids content of about 12%.

Considering the treated vegetation waters, with a content of totalsolids of about 7%, it was possible to concentrate by a factor of four,thus obtaining a permeate with a total solids content of 5.5% and aconcentrate with a total solids content of 12.1%.

The polyphenol concentrate of the present invention was produced byconcentrating the permeate obtained by microfiltering the vegetationwater by reverse osmosis.

Use was made of a polymeric membrane made of polyamide with a woundspiral shape, high salt rejection and a filtering surface of 7 m², butuse can also be made of membranes with low salt rejection and a minorloss of polyphenols in the permeate.

In reverse osmosis it is possible to concentrate the microfiltrationpermeate of the treated vegetation by a ratio of about 3.6.

Obviously, the volume concentration ratio can vary depending on theinitial starting matrix and above all its salt content and hence theosmotic pressure.

Composition of the Tested Vegetation Waters

The composition of the polyphenol concentrate is indicated in Table Ibelow:

TABLE I Phenolic Compounds Hydroxytyrosol 2.70 g/L Tyrosol 0.20 g/LChlorogenic acid 0.12 g/L β-hydroxyverbascoside isomer 1 0.35 g/Lβ-hydroxyverbascoside isomer 2 0.32 g/L Rutin 0.11 g/L Verbascoside 0.84g/L Luteolin-7-O-gluside 0.22 g/L 3,4-DHPEA-EDA 1.99 g/L The results areexpressed in g of tyrosol per L of water. Internal standard: SyringicAcid Metals Sodium 152 mg/L Calcium 2.95 g/L Magnesium 442 mg/LPotassium 7.6 g/L Anions Chlorides 3.4 g/L (expressed as NaCl) Sulphates22.78 g/L (expressed as K₂SO₄) Phosphates 3.2 g/L (expressed as PO₄ ³⁻)Nitrates 24.5 mg/L (expressed as nitric N) Carbohydrates Glucose 31 g/LFructose 7 g/L Mannitol 2 g/L Sucrose 8 g/L Nitrogen 0.03% 30 mg/kg

The substances shown in Table I are contained in the sample with theidentification code A009, whereas the blank (i.e. negative control) isidentified by the code A012.

The blank was obtained by batch chromatographic separation of thepolyphenol concentrate with XAD 7 resin.

In detail, a volume of about 50 cc of XAD 7 resin was rinsed withdistilled water, regenerated in ethanol and again rinsed with distilledwater.

The resin was recovered by vacuum filtration with a 0.45 micron filterand added to about 75 ml of concentrate of vegetation waters in abeaker. The resin was left in contact with the concentrate for about 30minutes under shaking at room temperature.

A vacuum filtration made it possible to recover the concentrate ofvegetation waters treated with XAD 7 resin with an electricalconductivity of 23.4 mS/cm.

The vegetation water concentrate recovered after treatment with XAD 7resin was again treated with XAD 7 resin, regenerated in ethanol andrinsed with distilled water.

After vacuum filtration, the third sample of the blank of vegetationwater concentrate treated twice with XAD 7 resin was recovered, with anelectrical conductivity of 16.92 mS/cm.

HUVECs (Human Umbelical Vein Endothelial Cells) were used as a model ofthe target cell of the polyphenol concentrate of the present invention,in consideration of the fact that the endothelial cell constitutes thefundamental unit of the process of angiogenesis.

The results obtained from the analyses conducted with the polyphenolconcentrate were compared with those obtained when treating the HUVECsunder the same conditions with hydroxytyrosol alone. Hydroxytyrosol waschosen as substance to make a comparison with as it is the polyphenolmost represented in sample A009 (2.70 g/L).

The aim is to demonstrate that vegetation water (in the form of aconcentrate as described), in addition to having an anti-angiogeniceffect, displays a better inhibitory ability than one substance alone.

In order to assess whether the effects were actually due to thehydroxytyrosol and not to the ethanol solution in which it wasdissolved, the analyses were performed using the medium containingethanol at the same concentration in which the hydroxytyrosol isdissolved as a blank sample.

Evaluation of the Anti-Angiogenic Properties of the Vegetation Waters

The dilutions of the samples correspond to values of mg/ml and μMpresent in Table II below:

TABLE II DILUTIONS mg/ml hydroxytyrosol μM hydroxytyrosol 1:100  0.0270174.96 1:250  0.0108 69.984 1:500  0.0054 34.992 1:1000 0.0027 17.4961:2500 0.00108 6.9984 1:5000 0.00054 3.4992  1:10000 0.00027 1.7496Evaluation of the Anti-Proliferative Effect of the Vegetation Waters onthe Endothelial Cells

The effect of samples A009 and A012 on the proliferation of the humanendothelial cells (HUVEC) was assessed by means of the MTT viabilityassay (tetrazoil salt,[3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide). Theassay is based on the ability of the MTT compound to be metabolized by amitochondrial enzyme, succinate dehydrogenase. The reduction of the saltleads to the formation of crystals of a blue-colored product, formazan,which is insoluble in water. The viable cells, unlike the non-viableones, reduce the salt and the amount of formazan produced isproportional to the number of cells present. The crystals formed aresolubilized and the absorbance values at the wavelength of 570 nm areread via the spectrophotometer.

5000 HUVE cells were seeded in each well of a 96-well plate. Inparticular, the wells were coated with 1% gelatin and the assays wereconducted at different treatment times (24 h, 48 h, 72 h, 96 h) and foreach time different dilutions of the concentrates were tested(1:10000-1:100 intervals).

The effect on endothelial cell proliferation was evaluated for thefollowing dilutions of concentrate:1:10000-1:5000-1:2500-1:1000-1:500-1:250-1:100.

As can be observed from FIG. 1A, there was a significant reduction inthe viability of the endothelial cells treated with sample A009, inparticular, starting from the 1:1000 dilution, after 24 h of treatment.

Sample A012 (the blank) had no effect on HUVEC viability (FIG. 1B).

Hydroxytyrosol also causes a reduction in the viability of HUVE cells,in particular starting from the 1:250 dilution, after 24 h of treatment(FIG. 1C). The medium containing ethanol, used as a control, does notproduce any effect on cell viability (FIG. 1D). The experiment wasconducted in duplicate and repeated twice.

It can thus be concluded, therefore, that sample A009 has a greatereffect on cell viability than hydroxytyrosol.

Evaluation of Apoptosis

The induction of apoptosis (programmed cellular death) of endothelialcells treated with the concentrate was evaluated by marking with annexinV and 7-Amino-actinomycin D (7-AAD). Cytofluorimetric analysis of thesemarkers makes it possible to distinguish, within the same cellpopulation, the ability of a treatment to induce cell death at differentstages (early, late, advanced). Annexin V is a molecule capable ofbinding phosphatidylserine, a glycerophospholipid of the cell membrane,normally associated with the internal cytosolic layer, which is exposedon the extracellular side as a result of cell damage. 7-AAD is acompound capable of passing through the cell membrane only in damagedcells or in apoptosis; positivity to this marker is therefore indicativeof cell death and toxicity of the product.

150,000 HUVE cells were seeded in a well of 6-well plate coated with 1%gelatin. The cells were subsequently treated (for 24 h and 48 h) withdifferent dilutions of the samples before being detached from the plateby treatment with trypsin and stained with 7-AAD and annexin V in orderto evaluate the levels of apoptosis.

The endothelial cells were treated for 24 and 48 hours at differentdilutions of samples A009, A012 and HyT-EtOH (range of dilutions1:2500-1:250) and analyzed for positivity to the two markers.

As can be noted from FIGS. 2A and 2C, a 1:250 dilution of sample A009induces the late stage of apoptosis in 50% and 75% of the endothelialcells, respectively, after 24 h and 48 h of treatment. The same dilutionof sample A012 does not exert any pro-apoptotic effect (about 95% of thecells are viable).

In contrast, the treatment with hydroxytyrosol and with the culturecontaining ethanol does not increase cellular necrosis (FIG. 3).

The experiment was conducted in duplicate and repeated twice.

Therefore, hydroxytyrosol displays less activity than the concentrate ofthe present invention.

Evaluation of the Effect of Vegetation Waters on the Morphogenesis ofEndothelial Cells

When they are cultured in vitro in an extracellular matrix and exposedto suitable activating stimuli, endothelial cells are capable oforganizing into tubular structures which mimic the structure of theinternal lumen of the vessels.

Via an assay of morphogenesis in matrigel (i.e. a polymer consisting oflaminin, collagen IV, entactin, heparan sulphate proteoglycan, growthfactors (e.g. PDGF, EGF, TGF-β and MMP) it is possible to evaluate theanti-angiogenic potential of selected compounds. In order to evaluatethe ability of the concentrate of the present invention to inhibit theformation of tubuli in vitro, each well of a 24-well plate was coatedwith 300 μL of matrigel (10 mg/mL) and, following polymerization of thematrix, 50,000 HUVE cells in 1 mL of complete medium were seeded in thepolymerized matrix. The cells were pre-treated for 24 hours withdifferent dilutions of samples A009, A012, HyT and EtOH (1:500 and1:250).

As positive and negative controls, use was made respectively of acomplete culture medium (CM) consisting of M199 medium supplemented with10 ng/ml of aFGF (acid Fibroblastic growth factor), 10 ng/ml of bFGF(basic Fibroblastic growth factor), 10 ng/ml of EGF (Epidermal growthfactor), 0.1 mg/ml of heparin, 0.10 μg/ml of hydrocortisone, 10% FBS(Fetal Bovine serum), 1% glutamin (Gin), 1% Ampicillin/Streptomycin(P/S) and the growth-factor and serum free medium M199 (SFM).

The inhibitory effect of the samples was evaluated after 6 hours ofincubation at 37° C. and 5% CO₂, by observation under a microscope ofthe formation of tubular structures.

As may be observed in FIG. 4A, sample A009 is capable of interferingwith the formation of stable tubular structures by the endothelial cellsin a dose-dependent manner. The same dilutions of sample A012 (blank) donot exert any inhibitory effect.

Hydroxytyrosol is also capable of interfering with the formation ofstructures stable tubular structures by the endothelial cells in adose-dependent manner, whereas the dilutions of ethanol (blank) exertonly a slight inhibitory effect (FIG. 4B). The experiment was conductedin duplicate and repeated twice.

Therefore, hydroxytyrosol inhibits endothelial morphogenesis with a lessmarked effect than sample A009, i.e. than the concentrate of the presentinvention.

Evaluation of the Inhibitory Potential of Vegetation Waters on theMigratory Activity of Endothelial Cells

Endothelial cells are characterized by the ability to migrate toward aspecific site, following a chemotactic gradient. In order to evaluatethe migration capacity of the HUVECs, migration assays were set up usingBoyden chambers and porous filters with a cut-off of 12 μm, soaked withcollagen (50 μg/mL).

The lower compartment of each chamber was loaded with 210 μL of CMcontaining 10 ng/mL aFGF, 10 ng/ml bFGF. 10 ng/mL EGF, 0.1 mg/mLheparin, 0.10 μg/mL hydrocortisone, 10% FBS, 1% Gln, 1% P/S orgrowth-factor and serum free medium M199 (SFM).

50000 HUVE cells pre-treated for 24 hours with diverse dilutions(1:2500, 1:1000, 1:500 and 1:250) of concentrate, of the blank, ofhydroxytyrosol or of the medium containing ethanol were seeded in theupper compartment of the chamber, in 500 μL of serum free medium.

The cells were incubated at 37° C., 5% CO₂ for 6 h.

At the end of the assay, the filters were mechanically cleaned so as toeliminate the non-migrating cells on the upper side; they were thenfixed in absolute ethanol for 5 minutes, and subsequently rehydrated andmarked with the viability stain DAPI. The cells present on each filterwere counted under a fluorescence microscope. In particular, 5 opticalfields per filter were analyzed in a random manner.

As shown in FIG. 5A, the concentrate of the present invention (sampleA009) is capable of interfering with the migratory capacity of theendothelial cells to a statistically significant degree at the 1:500 and1:250 dilutions (p-value=0.0057 and p-value=0.0003, respectively),whereas sample A012 (blank) does not interfere with the migratorycapacity of the same.

Hydroxytyrosol reduces the migratory capacity of the endothelial cellscompared to the medium with ethanol used at the 1:250 dilution with ap-value=0.0302 (FIG. 5B). It can also be observed that at higherdilutions of hydroxytyrosol the endothelial cells show an increase inmigratory capacity, whereas the concentrate of the present inventioninhibits it. The experiment was conducted in duplicate and repeatedtwice.

Therefore, hydroxytyrosol used as a single substance displays a lowerability to interfere with endothelial migration, i.e. it is noteffective.

Evaluation of the Inhibitory Potential of Vegetation Waters on theInvasive Activity of the Endothelial Cells

Once recruited in situ, endothelial cells have the ability to invade thesurrounding tissues, producing factors capable of degrading theextracellular matrix, which constitutes a physical barrier to theiractual migration to the chemotactic site.

The invasion assay was set up using Boyden chambers and porous filterswith a cut-off of 12 μm, coated with matrigel (1 mg/mL).

210 μL of complete culture medium (CM) containing 10 ng/mL aFGF, 10ng/ml bFGF, 10 ng/mL EGF, 0.1 mg/mL heparin, 0.10 μg/mL hydrocortisone,10% FBS, 1% Gln, 1% P/S or 210 μL of growth-factor and serum free mediumM199 (SFM) were poured into the lower compartment of each chamber.

50000 HUVE cells resuspended in 500 μL of SFM and pre-treated for 24hours with different dilutions (1:2500, 1:1000, 1:500 and 1:250) ofconcentrate, of the blank, of hydroxytyrosol and of the solution withouthydroxytyrosol were incubated at 37° C., 5% CO₂ for 24 h.

At the end of the assay, the filters were mechanically cleaned so as toeliminate the cells present on the upper side which had not penetratedbeyond the matrix. The filters containing the cells that had invaded thematrigel were fixed in absolute ethanol for 5 minutes, and thenrehydrated and marked with the viability stain DAPI. The cells presenton each filter were counted under a fluorescence microscope; 5 opticalfields per filter, selected in a random manner, were subjected toanalysis.

The results shown in FIG. 6A demonstrate that sample A009 (i.e. theconcentrate of the present invention) is capable of interfering with theinvasive capacity of the endothelial cells to a statisticallysignificant degree at the 1:500 and 1:250 dilutions (p-value=0.0335 andp-value=0.0011, respectively), whereas sample A012 (blank) does notinfluence the invasive capacity of the same.

As shown in FIG. 6B, hydroxytyrosol reduces the invasive capacity of theendothelial cells compared to the medium with ethanol used at the 1:500and 1:250 dilutions with a p-value=0.0016 and p-value=0.0159.

Hydroxytyrosol has better effectiveness considering longer times, sinceit inhibits cell invasion, but not migration.

The concentrate of the present invention functions effectively againstboth migration and invasion.

Evaluation of the Protective Role Against Damage Due to Oxidative Stressby the Vegetation Waters after Treatment with H₂O₂

Reactive Oxygen Species (ROS) represent one of the main mechanisms ofcellular damage and play a fundamental role in the inflammation processand, as a consequence, in the angiogenesis correlated with inflammation.An assessment was thus made of the antioxidant potential vis-à-vis HUVEcells subjected to treatment with the vegetation water concentratesafter treatment with H₂O₂, by marking with DCFH-DA(2′,7′-dichlorfluorescein-diacetate).

DCFH-DA is a substance capable of revealing the concentration ofintracellular H₂O₂ and can be detected by cytofluorimetry.

150,000 HUVE cells were seeded in complete medium in each well of aE-well plate coated with 1% gelatin. The cells were subsequentlytrypsinized and resuspended in complete M199 medium with H₂O₂ at aconcentration of 250 μM. The cells were then placed in an incubator at37° C. and 5% CO₂, in the dark, for 15 minutes. The cells weresubsequently washed with DPBS and the supernatant was then eliminated by5 minutes' centrifugation at 1200 rpm. Finally, the cells wereresuspended in DPBS containing 10 μM of DCFH-DA and H₂O₂ (positivecontrol) or dilutions of the concentrates (A009 and A012), ofhydroxytyrosol or of solvent free of hydroxytyrosol. The cells wereincubated at 37° C. in an atmosphere containing 5% CO₂ for 45 minutes inthe dark. Finally, the results were read using a FACSCanto.

The results summarized in FIG. 7A demonstrate that sample A009 (theconcentrate of the present invention) is capable of exerting aprotective effect in a dose-dependent manner and to a highly significantdegree: p-value 1:2500=0.0310, p-value 1:1000=0.0001, p-value1:500=0.0001, p-value 1:250<0.0001.

Hydroxytyrosol exerts an antioxidant effect (FIG. 7B), whereas themedium in which the ethanol is dissolved possesses no antioxidanteffect.

The results demonstrate that the concentrate of the present invention iscapable of exerting a more significant antioxidant effect. The p-valuesfor hydroxytyrosol are in fact lower: p-value 1:2500=0.0115, p-value1:1000=0.0062, p-value 1:500=0.0082 and p-value 1:250=0.0223.

The experiment was conducted in duplicate and repeated twice.

Evaluation of the Protective Role Against Damage Due to Oxidative Stressby the Vegetation Waters Before Treatment with H₂O₂

150,000 HUVE cells were seeded in complete medium in each well of aE-well plate coated with 1% gelatin. The cells were subsequentlytrypsinized and resuspended in complete M199 medium and the vegetationwater concentrates (A009 and A012), the hydroxytyrosol or mediumcontaining ethanol at the standard dilutions.

The cells were then placed in an incubator at 37° C. and 5% 002, in thedark, for 30 minutes. After washing with DPBS, the cells wereresuspended in DPBS with 10 μM of DCFH-DA and H₂O₂ both in the positivecontrol and in the samples under analysis. The cells were placed in anincubator at 37° C. and 5% CO₂ for 45 minutes in the dark. Finally, theresults were read using a FACSCanto.

In FIG. 8A it may be observed that sample A009 exerts a protectiveeffect in a dose-dependent manner, whilst it can be observed that theproduction of ROS remains high in the samples treated with the blank(A012). The reduction in the production of ROS is highly significant inthe case of sample A009: p-value 1:2500=0.0056, p-value 1:1000=0.0015,p-value 1:500<0.0001 and p-value 1:250<0.0001.

The results shown in FIG. 8B demonstrate that hydroxytyrosol has nosignificant antioxidant effect if used after induction of oxidation byH₂O₂. Furthermore, it is possible to observe that the solvent withouthydroxytyrosol produces no significant effect on the cells, as occurs inthe case of pre-treatment with H₂O₂.

The experiment was conducted in duplicate and repeated twice.

In conclusion, besides having a lower antioxidant power than theconcentrate of the present invention, hydroxytyrosol does not maintainthis potential if used in a pre-treatment after induction of oxidation.

Evaluation of the Anti-Angiogenic Potential of Vegetation Waters in aMouse Model

The ability of the vegetation water concentrate to inhibit blood vesselformation in vivo was evaluated by means of the matrigel sponge assay asdescribed by Albini et al. in “Angiogenic potential in vivo by Kaposi'ssarcoma cell-free supernatants and HIV-1 tat product: inhibition ofKS-like lesions by tissue inhibitor of metalloproteinase-2 (AIDS,1994)”.

Specifically, 8-week-old male mice of the strain c57/BL6 were used as ananimal model. Matrigel pellets were injected subcutaneously into themice, in association with sample A009, at different dilutions, or thecorresponding blank (A012). Matrigel appears in liquid form at atemperature of 4° C., and rapidly polymerizes at room temperature; thisproperty is exploited to simulate a subcutaneous tumor which secretespro-angiogenic substances. In this experiment, the matrigel in liquidform was associated with a mixture called VTH containing all of thefactors necessary for the process of angiogenesis, and consisting ofVEGF (100 ng/μl), TNF a (1.2 ng/μl) and heparin (25 U/ml).

VTH is considered a positive control, since VEGF represents the maingrowth factor for endothelial cells, TNFα constitutes a cytokine that isessential for recruiting the inflammatory cellular component and heparinserves to retain the blood that forms in the pellet as a result of therecruitment and activation of the endothelium. Once injectedsubcutaneously in the animal's flanks, matrigel polymerizes rapidly andreleases the factors contained in it. They act as a chemoattractant forthe endothelium and inflammatory cells which, once they have infiltratedthe matrigel, remain trapped and become activated in the“microenvironment” formed by the matrix. For the assay in question, 3mice were used for each condition. The conditions used were: 1) matrigelalone (negative control), 2) matrigel and VTH (positive control), 3)matrigel with VTH in combination with 2 dilutions of the vegetationwater concentrate (1:500 and 1:250), both for the sample under analysisand the blank sample. Moreover, the same dilutions were tested forhydroxytyrosol.

The matrigel pellets were injected on day 0; on day 4 the mice weresacrificed and the pellets were removed. The explanted pellets wereweighed and placed in 300 μl of PBS, and then divided into two parts:half was used for the assay of hemoglobin, which is an indicator ofangiogenesis (in the presence of blood in the pellet as a result of therecruitment of the endothelial cell and its activation), whereas theother half was embedded in OCT in order to carry out immunohistochemicalstaining to evaluate the endothelial and inflammatory component.

For the purpose of quantifying the hemoglobin, the pellets weremechanically disintegrated, centrifuged at 4° C. for 12 minutes at 13000g and the supernatant was removed.

200 μl of supernatant was then placed in an Eppendorf tube, where 800 μlof Drabkin solution were added. This substance binds to hemoglobin toform crystals which precipitate, thus enabling absorbance to be read bymeans of a spectrophotometer (540 nm): the hemoglobin concentration isdirectly proportional to the number of crystals formed and the recordedabsorbance. The quantification of hemoglobin relied on the followingmathematical model:HB=(absorbance at 540 nm/weight in mg of the pellet)×100

In FIG. 9A it is possible to observe that the staining is proportionalto the infiltration of the endothelium and consequent presence of blood.

In FIG. 9B it is possible to observe that the staining of the pellets ofthe 1:500 treatment with hydroxytyrosol is greater than that of thepositive control.

FIG. 10 shows the data related to quantification of the hemoglobincontent of the pellets. It can be noted that the hemoglobinconcentration decreases in the pellets associated with the concentrateof the present invention (A009), to a statistically significant degree(p-value=0.0058) compared to the pellets associated with sample A012(blank) and to hydroxytyrosol.

In conclusion, the experimental results reported above clearly indicatethat the polyphenol concentrate obtained from vegetation waters (sampleA009) is endowed with anti-angiogenic activity. In fact, the concentrateof the present invention inhibits the viability and increases theapoptosis of endothelial cells. Moreover, it also prevents the migrationand invasion of the synthetic matrices used to simulate theextracellular matrix. Finally, by treating the HUVECs with theconcentrate of the present invention it is also possible to observe theinhibition of the formation of the tubular structures of thevasculature.

In addition to the in vitro results, the anti-angiogenic potential ofthe concentrate of the present invention was clearly demonstrated alsoin a physiological system.

All of the experiments were conducted comparing the concentrate with thesame concentrate purified of the polyphenols (blank sample) so as to beable to affirm that the observed effects were due to the molecules ofinterest and not to other factors that might have interfered with theexperiments.

Furthermore, hydroxytyrosol was also tested on its own and the resultsof all the tests carried out demonstrated that it is less effective andactive than the concentrate of the present invention.

Therefore, the results of the experiments set forth above demonstratethat the polyphenol concentrate obtained by subjecting themicrofiltration permeate of vegetation waters to reverse osmosispossesses anti-angiogenic properties.

In particular, the concentrate has an improved anti-angiogenic effectcompared to hydroxytyrosol alone, which represents the main polyphenoliccompound contained in the vegetation waters.

The invention claimed is:
 1. A method of using a composition comprisinga concentrate of vegetation waters and/or olive pomace comprising 1-10g/L of hydroxytyrosol and 0.5-8 g/L of3,4-dihydroxyphenolethanol-elenolic acid di-aldehyde (3,4-DHPA-EDA) totreat and/or prevent inflammation, comprising the step of administeringthe composition to a patient in need thereof, wherein the compositiontreats and/or prevents inflammation, wherein the inflammation is a non-tumor inflammation selected from the group consisting of inflammatorydiseases of the colon-rectum, bronchial pathologies, liver diseases,diseases of the prostate, mucositis, and pre-neoplastic lesions.
 2. Themethod of claim 1, wherein the concentrate of the composition furthercomprises at least one phenolic compound selected from: tyrosol,chlorogenic acid, b-hydroxyverbascoside, rutin, verbascoside, andluteolin; and/or at least one metal selected from: sodium, calcium,magnesium and potassium; and/or at least an anion selected from:chlorides, sulphates, phosphates and nitrates; and/or at least onecarbohydrate selected from: glucose, fructose, mannitol and sucrose;and/or nitrogen.
 3. A method of using a beverage comprising aconcentrate composition of vegetation waters and/or olive pomacecomprising 1-10 g/L of hydroxytyrosol and 0.5-8 g/L of3,4-dihydroxyphenolethanol-elenolic acid di-aldehyde (3,4-DHPA-EDA) totreat and/or prevent inflammation, comprising the step of administeringthe beverage to a patient in need thereof, wherein the beverage treatsand/or prevents inflammation, wherein the inflammation is a non-tumorinflammation selected from the group consisting of inflammatory diseasesof the colon-rectum, bronchial pathologies, liver diseases, diseases ofthe prostate, mucositis, and pre-neoplastic lesions.
 4. The method ofclaim 3, wherein the beverage is based on water and fruits and/or milkand/or grape juice and/or must.